The neuropeptide Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (Neuropeptide FF or NPFF) modulates morphine analgesia, tolerance, and dependance (withdrawal or abstinence syndrome). Thus, NPFF studies have implications in treatment of drug addiction and chronic pain. This project's main thrust is to characterize NPFF receptors in mammalian CNS. We used a radioligand binding assay with 125I-YLFQPQRFa to determine the coupling and structure-activity relations (SAR) of NPFF receptors in membrane preparations of rat brain and spinal cord. We found that NPFF receptors are G-protein coupled (Gi or Go, not Gs) based on GTP regulation of NPFF receptor binding. We identified the optimal amino acids for receptor binding in each position of the C-terminal tetrapeptide portion of NPFF, and a pentapeptide designed as a photoaffinity ligand (D-YpbFnLRFa) had very high binding affinity. The proposed course of this project includes (I) identification of a cell line that expresses NPFF receptors, (2) investigation of the coupling of NPFF receptors to cellular signal transduction systems for development of an in vitro NPFF receptor bioassay, (3) development of specific NPFF antagonists, and (4) purification of NPFF receptors.